Lack of nifH gene in some endophytic bacteria isolated on RC solid medium

Presence of endophytic bacteria was reported in many crops including maize (Zea mays L.). Endophytes play a significant role in plant nutrient and pesticide uptake. Application of endophytic bacteria is a goal of sustainable agriculture. Occurrence of Azospirillum strains is often reported as tissue inhabiting bacteria of maize. The biological N2-fixation is one of most important processes assigned to this bacteria. The objective of this study was to examine the biodiversity of Azospirillum spp. isolated from the leaves of 6 cultivars of Zea mays L.. They were cultivated on two experimental fields at Smolice and Kobierzyce, (Poland). Strains of Azospirillum spp. were isolated on the solid RC medium. Forty four isolates grown as a small intensive red colonies were selected. To verify ability to N2-fixation isolates were analyzed based on nifH gene presence. Presence of nifH gene was tested using PCR method with PolF and PolR universal degenerate primers. The presence of nifH gene was found in 6 tested strains isolated from leaves of 3 cultivars (Cyrkon, Kosmo230, KB2704) from Smolice location, only. Our results suggest that selection of Azospirillum-like strains on RC solid medium based on appearance of colony is not correlated with theirs ability to nitrogen fixation or used degenerated primers (PolF, PolR) are not universal enough.     

高产吲哚乙酸及高泌氨巴西固氨螺菌的筛选与鉴定

目的：从玉米根际和土壤中分离具有高产吲哚乙酸较强的泌氨能力的巴西固氮螺菌。方法：分别通过半固体NFb培养基、CR培养基、LB培养基分离培养固氮菌株,并经过一系列菌落菌体形态特征、生理生化特性和16S rDNA序列测定等试验对其进行鉴定。结果：经分离纯化获得10株固氮菌,并鉴定均为巴西固氮螺菌（Azospirillum brasilense）,其中菌株R7在甘油半固体培养基上能分泌约14mmol/L的氨,在添加了色氨酸的培养基中能够合成58.8μg/ml的吲哚-3-乙酸（IAA）。结论：成功筛选得到一株既高产吲哚乙酸又有较强的泌氨能力的巴西固氮螺菌。     

A numerical taxonomic study of species of Vibrio isolated from the aquatic environment and birds in Kent, England

A numerical taxonomic study has been carried out to confirm the identity of strains of the family Vibrionaceae isolated during an ecological study. A total of 237 strains were studied including 148 from the aquatic environment, 6 from estuarine birds, 1 from sheep faeces, and 61 control cultures. Duplicates of 21 of the strains were randomly selected and included to estimate test and operator error. Taxonomic resemblance was estimated on the basis of 148 characters using Euclidean distance. The taxonomic position of some strains was reevaluated using the pattern different coefficient. Strains were clustered by three methods, all of which gave similar results. The estimated average probability of test error was 1.5%. Strains previously identified as Vibrio anguillarum fell into four distinct phenons corresponding to V. anguillarum biovar I, 'V. anguillarum biovar II', V. diazotrophicus, and strains pathogenic to oyster larvae. The latter group characteristically degraded xanthine and probably represents a new species. The phenon corresponding to V. cholerae included the type strain, strains of human origin, and strains isolated in the United Kingdom from birds and the aquatic environment. Some strains of V. cholerae were luminous. Other phenons were identified as V. metschnikovii, V. fluvialis, and Aeromonas spp.     

A numerical taxonomic study of species of Vibrio isolated from the aquatic environment and birds in Kent, England

A numerical taxonomic study has been carried out to confirm the identity of strains of the family Vibrionaceae isolated during an ecological study. A total of 237 strains were studied including 148 from the aquatic environment, 6 from estuarine birds, 1 from sheep faeces, and 61 control cultures. Duplicates of 21 of the strains were randomly selected and included to estimate test and operator error. Taxonomic resemblance was estimated on the basis of 148 characters using Euclidean distance. The taxonomic position of some strains was reevaluated using the pattern difference coefficient. Strains were clustered by three methods, all of which gave similar results. The estimated average probability of test error was 1.5%. Strains previously identified as Vibrio anguillarum fell into four distinct phenons corresponding to V. anguillarum biovar I, ' V. anguillarum biovar II', V. diazotrophicus , and strains pathogenic to oyster larvae. The latter group characteristically degraded xanthine and probably represents a new species. The phenon corresponding to V. cholerae included the type strain, strains of human origin, and strains isolated in the United Kingdom from birds and the aquatic environment. Some strains of V. cholerae were luminous. Other phenons were identified as V. metschnikovii, V. fluvialis , and Aeromonas spp.     

Leucobacter chromiireducens sp. nov, and Leucobacter aridicollis sp. nov., two new species isolated from a chromium contaminated environment

Two strains designated strains L-1^T and L-9^T were isolated from activated sludge of a treatment plant that receives wastewater from the tannery industry contaminated with chromium. Phylogenetic analysis showed that the organisms represented two new species of the genus Leucobacter. Strains L-1^T and L-9^T could be distinguished from the type strain of L. komagatae and from the type strain of “L. albus” by the B-type peptidoglycan composition, fatty acid composition, several phenotypic and physiological characteristics. The major fatty acids of the organisms were iso- and anteiso-branched C15:0 and C17:0, straight-chain C16:0 was also found in relatively high proportions. The organisms were halotolerant, grew in medium containing 9% NaCl, and all strains, including the type strain of L. komagatae grew in medium containing 5 mM Cr(VI). On the basis of the distinct peptidoglycan composition, 16S ribosomal DNA sequence analysis, percentage of DNA-DNA reassociation values, and phenotypic characteristics we are of the opinion that strain L-1^T represents a new species of the genus Leucobacter for which we propose the name Leucobacter chromiireducens and that strain L-9^T represents an additional new species of the same genus for which we propose the name Leucobacter aridicollis.     

Field inoculation of sorghum and rice withAzospirillum spp. andHerbaspirillum seropedicae

Two field experiments were carried out at the UAPNPBS experimental station, Seropédica, with two sorghum and one rice cultivars. The establishment, and inoculation effects, ofAzospirillum spp. andHerbaspirillum strains marked with antibiotic resistance were investigated. One grain sorghum (BR 300) and one sugar sorghum (Br 505) cultivar were used.Azospirillum lipoferum strain S82 (isolated from surface sterilized roots of sorghum) established in both cultivars and comprised 40 to 80% of theAzospirillum spp. population in roots and stems 60 days after plant emergence (DAE).Azospirillum amazonense strain AmS91 (isolated from surface-sterilized roots of sorghum) reached only 50%. At 90 DAE, S82 almost disappeared (less than 30% of establishment) while the establishment of AmS91 remained constant in roots and stems. No establishment ofH. seropedicae strain H25 (isolated from surface-sterilized roots of sorghum) orA. lipoferum strain S65 (isolated from the root surface of sorghum) could be observed on inoculated roots. Inoculation with S82, AmS91 or S65 but not withH. seropedicae H25, increased plant dry weight of both cultivars and total N in grain of the grain sorghum. In rice,A. lipoferum Al 121 andA. brasilense Sp 245 (isolated from surface sterilized rice and wheat roots respectively) established in the roots but there was no increase inAzospirillum spp. numbers due to inoculation. None of the strains affected plant growth or rice grain yield.Azospirillum amazonense, A82 andH. seropedicae Z95, which did not establish in roots, significantly enhanced seed germination.     

Determination of the structure of the repeated unit of the Azospirillum brasilense SR75 O-specific polysaccharide and homology of the lps loci in the plasmids of Azospirillum brasilense strains SR75 and Sp245

The structural identity of the repeated unit in O-specific polysaccharides (OPSs) present in the outer membrane of strain SR75 of the bacterium Azospirillum brasilense, isolated from wheat rhizosphere in Saratov oblast, and the OPSs of previously studied A. brasilense strain Sp245, isolated from surface-sterilized wheat roots in Brazil, has been demonstrated. Plasmid profiles, DNA restriction, and hybridization assays suggested that A. brasilense strains SR75 and Sp245 have different genomic structures. It was shown that homologous lps loci of both strains was localized in their plasmid DNA. This fact allows us to state that, despite their different origin, the development of the strains studied was convergent. Presumably, the habitation of these bacteria in similar ecological niches influenced this process in many respects.     

Oxoester oxidoreductase activities in new isolates of Pichia anomala from apple, grape and cane juices

Thirty-nine yeasts isolated from apple, grape and cane juices were screened for their oxidoreductase activity. The two strains of Pichia, one isolated from apple and one from cane juices, appear to be promising strains for oxidoreductase activity on alpha-oxoesters. They showed similar high yields in converting ethyl pyruvate to ethyl lactate as Saccharomyces spp. (86.6% and 85.3% versus 86.6%), and higher yields in the reduction of alpha-oxocarboxylic esters (ketopantolactone to pantolactone: 74% and 73.3%, respectively) compared to Saccharomyces spp. (yield 60%).     

Virulence factors in clinical and food isolates ofAeromonas species

Virulence factors were compared in 15Aeromonas spp. isolated from faeces of patients withAeromonas-associated gastroenteritis and in 81 strains isolated from food. Strains from food did not show differences in the distribution of virulence factors when compared with strains isolated from faeces. However, 88.8% ofAeromonas strains isolated from food were capable of producing possible virulence factors. Characterization of 28 autoagglutinating (AA⁺)Aeromonas spp. indicated that the human strains differed from the food strains in hemagglutinating and hemolytic capacities. These results suggest that auto-agglutination associated with hemagglutinating and hemolytic capacities in food strains may be a helpful indicator of potential pathogenicity.     

Nitrogenase activity (acetylene reduction) in root-associated, cold-climate species of Azospirillum, Enterobacter, Klebsiella and Pseudomonas growing at various temperatures

Abstract 23 Strains of diazotrophic root-associated bacteria isolated from various parts of Finland were tested for nitrogenase activity during growth at various temperatures. Nitrogenase activity was optimal at 20–37°C in cultures of Klebsiella pneumoniae , and at 14–20°C in cultures of Klebsiella terrigena and Enterobacter agglomerans . Strains of K. terrigena and E. agglomerans showed no activity at 37°C, and K. pneumoniae only minimal or no activity at 14°C. Azospirillum lipoferum exhibited high nitrogenase activity at both 28–37°C, but less than 25% of optimal activity at 20°C and no activity at 14°C. Pseudomonas sp. expressed nitrogenase activity at 14–28°C. None of the strains manifested nitrogenase activity at 4 or 42°C. There were only small local variations within a species between strains isolated at different locations.     

Occurrence, characterisation and detection of potential virulence determinants of emerging aquatic bacterial pathogens from the Philippines and Thailand

Strains of Aeromonas spp., 'non-cholera vibrios' (NCVs) and Plesiomonas shigelloides isolated from aquatic environments, fish and human diarrhoeal cases in the Philippines and Thailand were characterised for potential virulence markers. Thus, the production of cytotoxin, cell-associated and cell-free haemolysin and their capacity to adhere to human intestinal (Henle 407) cells in vitro was investigated. In addition, the occurrence of tlh and tdh haemolysin genes and urease activity among V. parahaemolyticus strains was investigated. The results showed that strains recovered from clinical sources (human and fish) produced these virulence factors, whereas these are absent in environmental strains.     

Selection of antagonists of postharvest apple parasites: Penicillium expansum and Botrytis cinerea

The objectives of this study were to constitute a collection of pathogenic agents of economic importance which cause losses of apple fruits after harvest namely Botrytis cinerea and Penicillium expansum and to select in vivo efficient antagonistic strains able to protect fruits against both pathogens at 5 degrees C (P. expansum) and 25 degrees C (P. expansum & B. cinerea). Twenty strains of P. expansum and ten strains of B. cinerea have been isolated from infected apple fruits. Potential antagonistic micro-organisms (thirty three isolates) belonging to yeast, bacteria and fungi have been isolated from apple surface. Six of them (strains Ach1-1, Ach2-1, Ach2-2 belonging to Aureobasidium pullulans (De Bary) Arnaud, and strains 1112-3, 1113-10 and 1113-5 belonging to Aureobasidium pullulans (de Bary) Am. v. pullulans) showed a high level of protection (more than 80%) at 25 degrees C. once inoculated with P. expansum or B. cinerea for 5 days. The highest level of protection against P. expansum (96%) was observed with the application of Ach 2-1. Six days after inoculation of B. cinerea, strains Ach 2-2 and Ach 2-1 insured 100% and 96% of protection, respectively. At lower temperature (5 degrees C), first symptoms of P. expansum appeared 13 days after its inoculation. Percentages of protection observed after apple treatment with one of the six antagonistic strains were ranged from 78% to 94% 20 days after P. expansum inoculation. Strains labelled Ach showed a protective level higher than 90% against this pathogen, followed by strain 1113-10 (90%), strain 1113-5 (89%) and strain 1112-3 (82%). At 26 days post-inoculation, levels of protection decreased but remained higher than 60% (more than 80% with strain Ach2-2 and strain 1113-5, 75% with strain Ach2-1 and 1113-10, 72% with ach1-1, 61% for the other strains). Strain Ach2-2 and 1113-10 were retained as the best antagonists for the subsequent studies.     

Characterization ofArxula adeninivorans strains from different habitats

SomeArxula adeninivorans strains selected from wood hydrolysates in Siberia, from soil in South Africa and from maize silage and soil in The Netherlands were compared. DNA-fingerprinting, pulse field gel electrophoresis as well as analysis of secretory proteins have been chosen to describe the similarities among the strains. Combination of the three methods allowed identification of each strain. Strains from the same origin show extensive similarities. The results of the DNA-fingerprints indicate that the strain isolated in Siberia belongs to the group of strains originated from South Africa. However, it differed in the molecular weight of the third chromosome and in the pattern of secretory proteins from the South African isolates.     

Isolation and characterization of nitrogen-fixing bacteria of the genus Azospirillum from the soil of a Sphagnum peat bog

The presence of nitrogen-fixing bacteria of the genus Azospirillum in the soils of acidic raised Sphagnum bogs is revealed for the first time. Three Azospirillum strains, B2, B21, and B22, were isolated as a component of methane-oxidizing enrichment cultures, whereas attempts to isolate them directly from peat samples have failed. The results of comparative analysis of the nucleotide sequences of 16S rRNA genes, DNA-DNA hybridization, and the analysis of the sequences of the functional genes encoding nitrogenase and ribulose-1, 5-bisphosphate carboxylase reveal that all the newly obtained strains can be classified as Azospirillum lipoferum. Yet, unlike A. lipoferum. the isolates do not require biotin and utilize sucrose, inositol, and glycerol for growth. The cell morphology of strain B2 differs from that of the type strain and strains B21 and B22. The results obtained indicate the variability of morphological, physiological, and biochemical properties in closely related Azospirillum strains and suggest the existence of metabolic relationships between methanotrophic bacteria and the representatives of the genus Azospirillum under peat bog conditions.     

Fatty acid analysis of four Azospirillum species reveals three groups

Cellular fatty acid composition of 14 strains from the four species of Azospirillum was determined by gas chromatographic analysis. All strains of Azospirillum lipoferum and Azospirillum brasilense were similar in fatty acid data, thus not revealing an expected distinction between the two long established species. Strains of both Azospirillum halopraeferens and Azospirillum amazonense, however, differed significantly from this first group of strains.     

Inhibition of Striga seed germination associated with sorghum growth promotion by soil bacteria

Striga spp. are obligate parasitic weeds of tropical cereals and generally have the same host range as rhizospheric bacteria of the genus Azospirillum. Four strains of Azospirillum brasilense, isolated from soil where sorghum is grown, have been tested for their effect on germination of Striga hermonthica seeds and on cereal (Sorghum vulgare) growth. Two out of four strains assayed significantly inhibited germination of the parasite. Moreover, one of the two strains showed a plant growth promoting (PGPR) effect.     

Characterization of six polymorphic microsatellite loci from Ampelomyces quisqualis,intracellular mycoparasite and biocontrol agent of powdery mildew

Ampelomyces spp. are common intracellular mycoparasites of powdery mildews worldwide and a strain has been commercialized as a biocontrol agent against these plant pathogens. In light of recent genetic analyses revealing high internal transcribed spacer sequence variability among Ampelomyces strains on different host plant mildews, yet no sequence variability within the Malus strain, polymorphic microsatellites were required to permit biocontrol and ecological studies of the complex apple/apple mildew/Ampellomyces tritrophic interaction. For this purpose, described here are the primers to amplify six polymorphic microsatellite loci from Ampelomyces quisqualis isolated from mycelia of the apple powdery mildew fungus, Podosphaera leucotricha.     

Characteristics of a pink-pigmented bacterium isolated from biofilm in a cooling tower in Tokyo, Japan

Strain K-20, a Gram-negative, non-motile, non-spore-forming and strictly aerobic rod, which produces a pale pink pigment, was isolated from biofilm in a cooling tower in Tokyo, Japan. The taxonomic feature of the strain was studied using phenotypic tests and phylogenetic analysis. Phylogenetic analysis of 16S rRNA gene sequences showed that the strain was related to Roseomonas gilardii subsp. rosea, Roseomonas gilardii subsp. gilardii, Roseomonas cervicalis and Roseomonas mucosa at 94.3-94.6 sequence similarities. Growth occurred at 25-40 C and pH 5.0-10.0, optimal at 35 C and pH 7.0. Growth did not occur in the presence of >or=2% NaCl. The API 20NE identification system gave a positive result for urease, L-arabinose, potassium gluconate, adipic acid, malic acid and trisodium citrate (API code number 0201465). The predominant fatty acids of strain K-20 were C18:1Delta11 (50.8%) and C16:1 (17.2%). Cells contained ubiquinone 10 (Q-10) as the major quinone and the G+C content was 72.0 mol%. Based on phenotypic, chemotaxonomic and phylogenetic data, it was assumed that strain K-20 (=JCM 14634) is a novel species of the genus Roseomonas.     

Cultured representatives of two major phylogroups of human colonic Faecalibacterium prausnitzii can utilize pectin, uronic acids, and host-derived substrates for growth

Faecalibacterium prausnitzii is one of the most abundant commensal bacteria in the healthy human large intestine, but information on genetic diversity and substrate utilization is limited. Here, we examine the phylogeny, phenotypic characteristics, and influence of gut environmental factors on growth of F. prausnitzii strains isolated from healthy subjects. Phylogenetic analysis based on the 16S rRNA sequences indicated that the cultured strains were representative of F. prausnitzii sequences detected by direct analysis of fecal DNA and separated the available isolates into two phylogroups. Most F. prausnitzii strains tested grew well under anaerobic conditions on apple pectin. Furthermore, F. prausnitzii strains competed successfully in coculture with two other abundant pectin-utilizing species, Bacteroides thetaiotaomicron and Eubacterium eligens, with apple pectin as substrate, suggesting that this species makes a contribution to pectin fermentation in the colon. Many F. prausnitzii isolates were able to utilize uronic acids for growth, an ability previously thought to be confined to Bacteroides spp. among human colonic anaerobes. Most strains grew on N-acetylglucosamine, demonstrating an ability to utilize host-derived substrates. All strains tested were bile sensitive, showing at least 80% growth inhibition in the presence of 0.5 μg/ml bile salts, while inhibition at mildly acidic pH was strain dependent. These attributes help to explain the abundance of F. prausnitzii in the colonic community but also suggest factors in the gut environment that may limit its distribution.     

Phenotypic heterogeneity of Pseudomonas corrugata strains from southern Italy

Fifty-five strains of Pseudomonas corrugata isolated in southern Italy were characterized phenotypically and compared with 23 strains of different origins. At least two main cultural types with rough or smooth colonies were observed. Strains with rough colonies produced a diffusible pigment in culture. On the basis of their nutritional profiles, Ps. corrugata strains formed a distinct phenon most closely related to fluorescent Pseudomonas spp. isolated from tomato pith necrosis-diseased plants. Three major groups of strains were differentiated within the Ps. corrugata phenon on the basis of utilization of 2-ketogluconate, meso-tartrate, hystamine, DL- glycerate and induction of a hypersensitive reaction on tobacco. Some Ps. corrugata strains belonging to group 1 and 3 which did not produce pigment in culture produced IAA in a colorimetric test. Variability in the serological reaction of the Italian strain was observed. None of the three antisera utilized reacted with all strains. Some strains isolated from diseased plants from the same greenhouse showed different nutritional profiles and reacted with different antisera. Fifteen lipopolysaccharide (LPS) patterns were observed. Strains were divided into two groups on the basis of their protein profiles. The heterogeneity which had already been observed in a world-wide study on Ps. corrugata was confirmed in strains from this restricted area.